| Aim and meaning of the studyMesenchymal stem cells (MSCs) originated from mesoblast's adult stem cells with high self-renewal capacity and multi-directional differentiation potency. MSCs could not only differentiate towards many tissues and cells, but also are easy to separate, culture and amplify. At the same time, exogenous genes are easy to be introduced and express in MSCs. During the course of cultivation for long term, the cells could remain multi-directional differentiation potency all the time and genetic background was much stabilized. At present, clinic tests had testified that transplantation of MSCs could be applied in recovering damage and curing genetic defect disease from mesenchymal tissue. So its prospect in clinic is very wide. Because of the property of MSCs, they were used in recovering damage, cell succedaneous cure, supporting haematogenesis and gene cure.MSCs are mesoblast's stem cells. In theory, they can differentiate into all types of cells in mesoblast, and they could also exist in all tissues coming from mesoblast. According to theory and plenty of experiments, base cells of any organ and tissue are all their sustaining cells. So, there are interstitial cell linespossessing the properties of stem cell in many organs and tissues. Many studies have testified that there are MSCs in tissues with active cell update system in vivo. At present, MSCs are testified to exist in bone marrow, liver, spleen, muscle, brain and skin. Although it is uncertain whether safer tissues contain MSCs, the work needs more studies and extensive investigation. Pancreas is a kind of stable tissue. Hu Ying etc testified that there were MSCs in fetal pancreas. The presence of this kind of cells provides a new direction for cell cure for pancreas disease and impairment. If in vitro, pancreases'MSCs could be separated and cultured successfully and be used to cure disease related to pancreas, such as diabetes, it will be meaningful to people's health. The aim of our study is to establish a separation-culture- purification way of MSCs originating from pancreas in vitro, testify their stem cells properties and potentiality in induction and differentiation and offer base achievement for cell source in cell transplantation. Besides, by recovery experiment in animals, the effect mechanism of MSCs could be elucidated preliminarily settled a base for curing diabetes.Methods1 To separate, culture and purify MSCs of neonate ratPancreas of neonate rat was digested to get cells, and then purified the cells to get pancreas MSCs by the way of adherence-digestion-passage-adherence. To identify its property of stem cell, morphological observation, determination of surface antigen, growth cycle and growth curve were applied. Besides, compare it with B-MSC that has been studied more at present.1.1 identification of differentiation potencyAt the base of normal culture, put antileptic of adult bone and lipoids intopurified cells, observe cellular reaction to antileptic and testify cellular origin and diverse differentiation potency.1.2 exploration on the function of repairing damageThe way of ligation was applied to establish the model of acute ischemic and necrotic rat pancreas. The purified cells labeled by fluorescence were transplanted to the rest normal part of the pancreas. Next, observe if labeled cells could migrate to impairment tissue and recover it.Result1 The way of separation and amplification in vitroThe cells separating from pancreas of neonate rat were cultured in culture flask at 105/ml. It could be found that cellular there were big difference between appearance in primary cells. Some were fusiform, some were round and polygon , big and cloning growth. But cells got to coincidence in appearance after passage and most of them were fusiform. FCM was applied to determine surface antigen CD34,CD44 of MSCs, but the value was very small and there was no obvious difference compared with bone marrow MSCs. So it testified that what we got were not tissue parenchymatous cells but undifferentiated immature cells in pancreas. According to growth curve, the mean doubling time of pancreas MSCs were about 5 days that means cells have high multiplication ability, so the number of cells for clinic application could be guaranteed. According to the growth cycle of the third generation, 90.76 % cells were the phase of G0-G1, 7.18 % were G2-M and 2.06 % were S that means caryocinesia is high in MSCs and multiplication ability is active. This kind of MSCs have higher doubling differentiation potency compared with normal general adult cells and they have important meaning for recoveringdamage.2 To testify potency of diverse differentiationThe cellular appearance changed obviously after being induced by osteoblast. Ancipital and random cells accumulated. Obvious calcification nodus could be observed under light microscope. At this time, cellular alinement was kind of directionality, and it showed whirlpool growth. In the middle of the whirlpool, cells showed multilamellar distribution, cellular limit wasn't distinct and most of cells were polygon or round. calfserum. The derived cells were stained by histochemical staining of Von Kossa. It was not obvious on the tenth day, but on the 20th day, grey and black particle could be found in positive cells'endochylema. After ALP staining, brown or black particle could be observed in endochylema and the color was thick or light, but in intensive region the color was thicker.The cells cultured in adult lipoids inducer changed gradually to round, cellular membrane was thin and the circumsciption of cellular membrane was not smooth. Fat little drops with high refraction could be found near nucleus after 3 days or so. Fat drops got more gradually and fused to big fat drops after 7 days or so, then changed to fat vacuole. Cells changed from fusiform to round or polygon. Most of big fat drops were round, inequality of size and didn't aggregate. Cellular nucleus remained at edge. Fat drops changed to orange after staining. Fat cells contained fat drops of different size and cellular nucleus, which showed blue after hematoxylin staining, was squeezed to one side.3 To testify the function of recovering damageAfter ligating pancreas for 7-8h, obvious liquid could be found whenopening abdominal cavity. The tail of pancreas ligated was black. According to pathological blade, a great quantity inflamed cells infiltrated into pancreas surrounding, islet cell disintegrated and pancreas got necrosis topically. After 2 weeks of transplantation, in experimental groups imbedding pancreas MSC, 15 rats survived and survival rate was 75%. Necrotic pancreas tissue recovered mainly to normal in naked eyes and the same in tissue pathological blade, but few inflamed cells could be found near blood vessel. Blue cells labeled by DAPI could be observed under fluorescence microscope. In contrast group, 4 rats survived and the survival rate was 20%. There was obvious difference between experimental groups and contrast group(p﹤0.05).ConclusionThe results of the study showed that there were MSC proliferating fast and diverse differentiations in pancreas. The cells could be cultured and amplified in vitro. To transplant them into damaged parts of pancreas in rats at the same race could promote obviously the recovery of damage and increase survival rate. Besides, there was great number of donator cells in damaged tissue that testified that transplanting donator cells has the great possibility of advancing recovery damage. Of course, whether donator pancreas MSC transformed to pancreas parenchymal cell or secreting recovery factors was not certain.
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